Genome sequence of the plant pathogen Ralstonia solanacearum.

Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. It is a model system for the dissection of molecular determinants governing pathogenicity. We present here the complete genome sequence and its analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. Both replicons have a mosaic structure providing evidence for the acquisition of genes through horizontal gene transfer. Regions containing genetically mobile elements associated with the percentage of G+C bias may have an important function in genome evolution. The genome encodes many proteins potentially associated with a role in pathogenicity. In particular, many putative attachment factors were identified. The complete repertoire of type III secreted effector proteins can be studied. Over 40 candidates were identified. Comparison with other genomes suggests that bacterial plant pathogens and animal pathogens harbour distinct arrays of specialized type III-dependent effectors.

A physical map of human chromosome 14.

We report the construction of a tiling path of around 650 clones covering more than 99% of human chromosome 14. Clone overlap information to assemble the map was derived by comparing fully sequenced clones with a database of clone end sequences (sequence tag connector strategy). We selected homogeneously distributed seed points using an auxiliary high-resolution radiation hybrid map comprising 1,895 distinct positions. The high long-range continuity and low redundancy of the tiling path indicates that the sequence tag connector approach compares favourably with alternative mapping strategies.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

Selfish DNA in protein-coding genes of Rickettsia.

Rickettsia conorii, the aetiological agent of Mediterranean spotted fever, is an intracellular bacterium transmitted by ticks. Preliminary analyses of the nearly complete genome sequence of R. conorii have revealed 44 occurrences of a previously undescribed palindromic repeat (150 base pairs long) throughout the genome. Unexpectedly, this repeat was found inserted in-frame within 19 different R. conorii open reading frames likely to encode functional proteins. We found the same repeat in proteins of other Rickettsia species. The finding of a mobile element inserted in many unrelated genes suggests the potential role of selfish DNA in the creation of new protein sequences.

Genomic exploration of the hemiascomycetous yeasts: 1. A set of yeast species for molecular evolution studies.

The identification of molecular evolutionary mechanisms in eukaryotes is approached by a comparative genomics study of a homogeneous group of species classified as Hemiascomycetes. This group includes Saccharomyces cerevisiae, the first eukaryotic genome entirely sequenced, back in 1996. A random sequencing analysis has been performed on 13 different species sharing a small genome size and a low frequency of introns. Detailed information is provided in the 20 following papers. Additional tables available on websites describe the ca. 20000 newly identified genes. This wealth of data, so far unique among eukaryotes, allowed us to examine the conservation of chromosome maps, to identify the 'yeast-specific' genes, and to review the distribution of gene families into functional classes. This project conducted by a network of seven French laboratories has been designated 'Génolevures'.

Genomic exploration of the hemiascomycetous yeasts: 2. Data generation and processing.

The generation of sequencing data for the hemiascomycetous yeast random sequence tag project was performed using the procedures established at GENOSCOPE. These procedures include a series of protocols for the sequencing reactions, using infra-red labelled primers, performed on both ends of the plasmid inserts in the same reaction tube, and their analysis on automated DNA sequencers. They also include a package of computer programs aimed at detecting potential assignation errors, selecting good quality sequences and estimating their useful length.

Genomic exploration of the hemiascomycetous yeasts: 5. Saccharomyces bayanus var. uvarum.

Saccharomyces bayanus var. uvarum investigated here is the species closest to Saccharomyces cerevisiae. Random sequence tags (RSTs) allowed us to identify homologues to 2789 open reading frames (ORFs) in S. cerevisiae, ORFs duplicated in S. uvarum but not in S. cerevisiae, centromeres, tRNAs, homologues of Ty1/2 and Ty4 retrotransposons, and a complete rDNA repeat. Only 13 RSTs seem to be homologous to sequences in other organisms but not in S. cerevisiae. As the synteny between the two species is very high, cases in which synteny is lost suggest special mechanisms of genome evolution. The corresponding RSTs revealed that S. uvarum can exist without any S. cerevisiae DNA introgression. Accession numbers are from AL397139 to AL402278 in the EMBL databank.

Genomic exploration of the hemiascomycetous yeasts: 6. Saccharomyces exiguus.

Random sequence tags were obtained from a genomic DNA library of Saccharomyces exiguus. The mitochondrial genome appeared to be at least 25.7 kb in size, with a different organization compared to Saccharomyces cerevisiae. An unusual putative 953 bp long terminal repeated element associated to Ty3 was found. A set of 1451 genes was identified homologous to S. cerevisiae open reading frames. Only five genes were identified outside the S. cerevisiae taxon, confirming that S. exiguus is phylogenetically closely related to S. cerevisiae. Unexpectedly, numerous duplicated genes were found whereas they are unique in S. cerevisiae. The sequences are deposited at EMBL under the accession numbers: AL407377-AL409955.

Genomic exploration of the hemiascomycetous yeasts: 7. Saccharomyces servazzii.

The genome of Saccharomyces servazzii was analyzed with 2570 random sequence tags totalling 2.3 Mb. BLASTX comparisons revealed a minimum of 1420 putative open reading frames with significant homology to Saccharomyces cerevisiae (58% aa identity on average), two with Schizosaccharomyces pombe and one with a human protein, confirming that S. servazzii is closely related to S. cerevisiae. About 25% of the S. servazzii genes were identified, assuming that the gene complement is identical in both yeasts. S. servazzii carries very few transposable elements related to Ty elements in S. cerevisiae. Most of the mitochondrial genes were identified in eight contigs altogether spanning 25 kb for a predicted size of 29 kb. A significant match with the Kluyveromyces lactis linear DNA plasmid pGKL-1 encoded RF4 killer protein suggests that a related plasmid exists in S. servazzii. The sequences have been deposited with EMBL under the accession numbers AL402279-AL404848.

Genomic exploration of the hemiascomycetous yeasts: 8. Zygosaccharomyces rouxii.

This paper reports the genomic analysis of strain CBS732 of Zygosaccharomyces rouxii, a homothallic diploid yeast. We explored the sequences of 4934 random sequencing tags of about 1 kb in size and compared them to the Saccharomyces cerevisiae gene products. Approximately 2250 nuclear genes, 57 tRNAs, the rDNA locus, the endogenous pSR1 plasmid and 15 mitochondrial genes were identified. According to 18S and 25S rRNA cladograms and to synteny analysis, Z. rouxii could be placed among the S. cerevisiae sensu lato yeasts.

Genomic exploration of the hemiascomycetous yeasts: 9. Saccharomyces kluyveri.

The genome of Saccharomyces kluyveri was explored through 2528 random sequence tags with an average length of 981 bp. The complete nuclear ribosomal DNA unit was found to be 8656 bp in length. Sequences homologous to retroelements of the gypsy and copia types were identified as well as numerous solo long terminal repeats. We identified at least 1406 genes homologous to Saccharomyces cerevisiae open reading frames, with on average 58.1% and 72.4% amino acid identity and similarity, respectively. In addition, by comparison with completely sequenced genomes and the SwissProt database, we found 27 novel S. kluyveri genes. Most of these genes belong to pathways or have functions absent from S. cerevisiae, such as the catabolic pathway of purines or pyrimidines, melibiose fermentation, sorbitol utilization, or degradation of pollutants. The sequences are deposited in EMBL under the accession numbers AL404849-AL407376.

Genomic exploration of the hemiascomycetous yeasts: 10. Kluyveromyces thermotolerans.

A genomic exploration of Kluyveromyces thermotolerans was performed by random sequence tag (RST) analysis. We sequenced 2653 RSTs corresponding to inserts sequenced from both ends. We performed a systematic comparison with a complete set of proteins from Saccharomyces cerevisiae, other completely sequenced genomes and SwissProt. We identified six mitochondrial genes and 1358-1496 nuclear genes by comparison with S. cerevisiae. In addition, 25 genes were identified by comparison with other organisms. This corresponds to about 24% of the estimated gene content of this organism. A lower level of conservation is observed with orthologues to genes of S. cerevisiae previously classified as orphans. Gene order was found to be conserved between S. cerevisiae and K. thermotolerans in 56.5% of studied cases.

Genomic exploration of the hemiascomycetous yeasts: 11. Kluyveromyces lactis.

Random sequencing of the Kluyveromyces lactis genome allowed the identification of 2235-2601 open reading frames (ORFs) homologous to S. cerevisiae ORFs, 51 ORFs which were homologous to genes from other species, 64 tRNAs, the complete rDNA repeat, and a few Ty1- and Ty2-like sequences. In addition, the complete sequence of plasmid pKD1 and a large coverage of the mitochondrial genome were obtained. The global distribution into general functional categories found in Saccharomyces cerevisiae and as defined by MIPS is well conserved in K. lactis. However, detailed examination of certain subcategories revealed a small excess of genes involved in amino acid metabolism in K. lactis. The sequences are deposited at EMBL under the accession numbers AL424881-AL430960.

Genomic exploration of the hemiascomycetous yeasts: 12. Kluyveromyces marxianus var. marxianus.

As part of the comparative genomics project 'GENOLEVURES', we studied the Kluyveromyces marxianus var. marxianus strain CBS712 using a partial random sequencing strategy. With a 0.2 x genome equivalent coverage, we identified ca. 1300 novel genes encoding proteins, some containing spliceosomal introns with consensus splice sites identical to those of Saccharomyces cerevisiae, 28 tRNA genes, the whole rDNA repeat, and retrotransposons of the Ty1/2 family of S. cerevisiae with diverged Long Terminal Repeats. Functional classification of the K. marxianus genes, as well as the analysis of the paralogous gene families revealed few differences with respect to S. cerevisiae. Only 42 K. marxianus identified genes are without detectable homolog in the baker's yeast. However, we identified several genetic rearrangements between these two yeast species.

Genomic exploration of the hemiascomycetous yeasts: 13. Pichia angusta.

As part of a comparative genomics project on 13 hemiascomycetous yeasts, the Pichia angusta type strain was studied using a partial random sequencing strategy. With coverage of 0.5 genome equivalents, about 2500 novel protein-coding genes were identified, probably corresponding to more than half of the P. angusta protein-coding genes, 6% of which do not have homologs in Saccharomyces cerevisiae. Some of them contain one or two introns, on average three times shorter than those in S. cerevisiae. We also identified 28 tRNA genes, a few retrotransposons similar to Ty5 of S. cerevisiae, solo long terminal repeats, the whole ribosomal DNA cluster, and segments of mitochondrial DNA. The P. angusta sequences were deposited in EMBL under the accession numbers AL430961 to AL436044.

Genomic exploration of the hemiascomycetous yeasts: 14. Debaryomyces hansenii var. hansenii.

By analyzing 2830 random sequence tags (RSTs), totalling 2.7 Mb, we explored the genome of the marine, osmo- and halotolerant yeast, Debaryomyces hansenii. A contig 29 kb in length harbors the entire mitochondrial genome. The genes encoding Cox1, Cox2, Cox3, Cob, Atp6, Atp8, Atp9, several subunits of the NADH dehydrogenase complex 1 and 11 tRNAs were unambiguously identified. An equivalent number of putative transposable elements compared to Saccharomyces cerevisiae were detected, the majority of which are more related to higher eukaryote copia elements. BLASTX comparisons of RSTs with databases revealed at least 1119 putative open reading frames with homology to S. cerevisiae and 49 to other genomes. Specific functions, including transport of metabolites, are clearly over-represented in D. hansenii compared to S. cerevisiae, consistent with the observed difference in physiology of the two species. The sequences have been deposited with EMBL under the accession numbers AL436045-AL438874.

Genomic exploration of the hemiascomycetous yeasts: 15. Pichia sorbitophila.

This paper reports the genomic analysis of the strain CBS7064 of Pichia sorbitophila, a homothallic diploid yeast. We sequenced 4829 random sequence tags of about 1 kb and compared them to the Saccharomyces cerevisiae gene products. Approximately 1300 nuclear genes, 22 tRNAs, the rDNA locus, and six mitochondrial genes have been identified. The analysis of the rDNA genes has permitted to classify this organism close to the Candida species. Accession numbers from AL414896 to AL419724 at EMBL databank.

Genomic exploration of the hemiascomycetous yeasts: 16. Candida tropicalis.

The genome of the diploid hemiascomycetous yeast Candida tropicalis, an opportunistic human pathogen and an important organism for industrial applications, was explored by the analysis of 2541 Random Sequenced Tags (RSTs) covering about 20% of its genome. Comparison of these sequences with Saccharomyces cerevisiae and other species permitted the identification and the analysis of a total of more than 1000 novel genetic elements of C. tropicalis. Moreover, the present study confirms that in C. tropicalis, the rare CUG codon is read as a serine and not a leucine. The sequences have been deposited at EMBL with the accession numbers AL438875-AL441602.